Manipulating chromatin architecture in C. elegans

Abstract

Background: Nucleosome-mediated chromatin compaction has a direct effect on the accessibility of trans-acting activators and repressors to DNA targets and serves as a primary regulatory agent of genetic expression. Understanding the nature and dynamics of chromatin is fundamental to elucidating the mechanisms and factors that epigenetically regulate gene expression. Previous work has shown that there are three types of canonical sequences that strongly regulate nucleosome positioning and thus chromatin accessibility: putative nucleosome-positioning elements, putative nucleosome-repelling sequences, and homopolymeric runs of A/T. It is postulated that these elements can be used to remodel chromatin in C. elegans. Here we show the utility of such elements in vivo, and the extreme efficacy of a newly discovered repelling sequence, PRS-322.

Results: In this work, we show that it is possible to manipulate nucleosome positioning in C. elegans solely using canonical and putative positioning sequences. We have not only tested previously described sequences such as the Widom 601, but also have tested additional nucleosome-positioning sequences: the Trifonov sequence, putative repelling sequence-322 (PRS-322), and various homopolymeric runs of A and T nucleotides.

Conclusions: Using each of these types of putative nucleosome-positioning sequences, we demonstrate their ability to alter the nucleosome profile in C. elegans as evidenced by altered nucleosome occupancy and positioning in vivo. Additionally, we show the effect that PRS-322 has on nucleosome-repelling and chromatin remodeling.

Keywords: C. elegans; Chromatin; Epigenetics; Nucleosome occupancy; Nucleosome positioning; Nucleosomes; PRS-322; Transgene; Widom 601.

Fig. 1

Sequences of putative positioning (601 and Trifonov), and putative repelling [PRS-322, poly(A) and poly(T) sequences and de Boer-derived sequences] DNA elements with dinucleotide or longer runs of As or Ts highlighted in red

Fig. 2

Depiction of transgene constructs. pBYU1(the control construct) with the unc-54 minimal enhancer (blue). pBYU9, pBYU14, and pBYU18 have putative nucleosome positioning [601(orange) or Trifonov (purple)] elements around the unc-54 minimal enhancer. pBYU28, pBYU29, pBYU41, and pBYU44 were used to test putative repelling elements; pBYU28 with 20 bp poly-A repeats (red), pBYU29 with the 70 bp PRS-322 sequence (black), pBYU41 with forward- and reverse-oriented 5 bp poly-T sequences (turquoise), and pBYU44 with an AAAAAcAAAAA sequence (dark blue) downstream of the unc-54 minimal enhancer (blue)

Fig. 3

Nucleosome occupancy, the overall number of nucleosomes bound to DNA at a given locus. In all constructs the unc-54 minimal enhancer locus is present and highlighted in blue with the enhancer center designated as the origin and the x-axis numbering in base pairs. The y-axis is the normalized nucleosome occupancy. A pBYU1, with only the unc-54 minimal enhancer locus. b pBYU9 with the Trifonov sequence loci highlighted in purple. C pBYU14 with the 601 sequence locus in orange. D pBYU18 with the two 601 sequence loci in orange. E pBYU29 with the two 70-bp PRS-322 loci in gray. F pBYU28 with the two 20-bp poly-A runs highlighted in red. G pBYU41 with the forward and reverse 5-bp poly-T runs in light blue. H pBYU44 with the single AAAAAcAAAAA sequence locus in dark blue

Fig. 4

Comparative nucleosome occupancy of regions flanking the unc-54 enhancer and inserted elements. A Nucleosome occupancy for the flanking regions for pBYU1 (dashed black) compared to the flanking regions from transgenes with positioning elements: pBYU9 (red), pBYU14 (blue), pBYU18 (green). B Nucleosome occupancy for the flanking regions for pBYU1 (dashed black) compared to the flanking regions from transgenes with repelling elements: pBYU28 (purple), pBYU29 (orange). C Nucleosome occupancy for the flanking regions for pBYU1 (dashed black) compared to the flanking regions from transgenes with repelling elements derived from the de Boer analysis: pBYU41 (brown), pBYU44 (yellow). For all three graphs, the unc-54 enhancer and the inserted positioning/repelling sequences have been removed with position 0 being where those elements would have been located. Negative x-axis values are the number of base pairs upstream, and positive X-axis values are the number of base pairs downstream of these elements

Fig. 5

Nucleosome positioning, the percent of nucleosomes (dyads) positioned within a 21-bp window surrounding a locus compared to all the nucleosomes (dyads) within the 301-bp window surrounding that same locus. As in Fig. 3, in all constructs, the unc-54 minimal enhancer locus is present and highlighted in blue with the enhancer center designated as the origin and the x-axis numbering in base pairs. The y-axis is the percent nucleosome positioning score represented as a decimal. A pBYU1, with only the unc-54 minimal enhancer locus. B pBYU9 with the Trifonov sequence loci highlighted in purple. C pBYU14 with the 601 sequence locus in orange. D pBYU18 with the two 601 sequence loci in orange. E pBYU29 with the two 70 bp PRS-322 loci in gray. F pBYU28 with the two 20 bp poly-A runs highlighted in red. G pBYU41 with the forward and reverse 5 bp poly-T runs in light blue. H pBYU44 with the single AAAAAcAAAAA sequence locus in dark blue

Fig. 6

Plasmid map of pPD151.79